Laser capture microdissection translation stage joystick

ABSTRACT

Systems and methods for laser capture microdissection are disclosed. An inverted microscope includes a translation stage joystick subsystem. The systems and methods provide the advantages of increased speed and much lower rates of contamination.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is, under 35 U.S.C. §120, a continuation of U.S.Ser. No. 09/018,452, filed Feb. 4, 1998, now pending, which is in-turn acontinuation-in-part of both U.S. Ser. No. 60/060,731, filed Oct. 1,1997, now pending, and U.S. Ser. No. 60/037,864, filed Feb. 7, 1997, nowabandoned, the entire contents of all which are hereby incorporatedherein by reference as if fully set forth herein.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention relates generally to the field of laser capturemicrodisection (LCM). More particularly, the invention relates toinverted microscopes that include specialized apparatus for performingLCM. Specifically, a preferred implementation of the invention relatesto an inverted microscope that includes a cap handling subsystem, anillumination/laser optical subsystem, a vacuum chuck subsystem, and amanual joystick subsystem. The invention thus relates to invertedmicroscopes of the type that can be termed laser capture microdisectioninverted microscopes.

[0004] 2. Discussion of the Related Art

[0005] Diseases such as cancer have long been identified by examiningtissue biopsies to identify unusual cells. The problem has been thatthere has been no satisfactory prior-art method to extract the cells ofinterest from the surrounding tissue. Currently, investigators mustattempt to manually extract, or microdissect, cells of interest eitherby attempting to mechanically isolate them with a manual tool or througha convoluted process of isolating and culturing the cells. Mostinvestigators consider both approaches to be tedious, time-consuming,and inefficient.

[0006] A new technique has been developed which can extract a smallcluster of cells from a tissue sample in a matter of seconds. Thetechnique is called laser capture microdissection (LCM). Laser capturemicrodissection is a one-step technique which integrates a standardlaboratory microscope with a low-energy laser and a transparent ethylenevinyl acetate polymer thermoplastic film such as is used for the plasticseal in food product packaging.

[0007] In laser capture microdissection, the operator looks through amicroscope at a tissue biopsy section mounted on a standard glasshistopathology slide, which typically contains groups of different typesof cells. A thermoplastic film is placed over and in contact with thetissue biopsy section. Upon identifying a group of cells of interestwithin the tissue section, the operator centers them in a target area ofthe microscope field and then generates a pulse from a laser such as acarbon dioxide laser having an intensity of about 50 milliwatts (mW) anda pulse duration of between about 50 to about 500 milliseconds (mS). Thelaser pulse causes localized heating of the plastic film as it passesthrough it, imparting to it an adhesive property. The cells then stickto the localized adhesive area of the plastic tape directly above them,whereupon the cells are immediately extracted and ready for analysis.Because of the small diameter of the laser beam, extremely small cellclusters may be microdissected from a tissue section.

[0008] By taking only these target cells directly from the tissuesample, scientists can immediately analyze the gene and enzyme activityof the target cells using other research tools. Such procedures aspolymerase chain reaction amplification of DNA and RNA, and enzymerecovery from the tissue sample have been demonstrated. No limitationshave been reported in the ability to amplify DNA or RNA from tumor cellsextracted with laser capture microdissection.

[0009] Laser capture microdissection has successfully extracted cells inall tissues in which it has been tested. These include kidney glomeruli,in situ breast carcinoma, atypical ductal hyperplasia of the breast,prostatic interepithielial neoplasia, and lymphoid follicles. The directaccess to cells provided by laser capture microdissection will likelylead to a revolution in the understanding of the molecular basis ofcancer and other diseases, helping to lay the groundwork for earlier andmore precise disease detection.

[0010] Another likely role for the technique is in recording thepatterns of gene expression in various cell types, an emerging issue inmedical research. For instance, the National Cancer Institute's CancerGenome Anatomy Project (CGAP) is attempting to define the patterns ofgene expression in normal, precancerous, and malignant cells. Inprojects such as CGAP, laser capture microdissection is a valuable toolfor procuring pure cell samples from tissue samples.

[0011] The LCM technique is generally described in the recentlypublished article: Laser Capture Microdissection, Science, Volume 274,Number 5289, Issue 8, pp 998-1001, published in 1996, the entirecontents of which are incorporated herein by reference. The purpose ofthe LCM technique is to provide a simple method for the procurement ofselected human cells from a heterogeneous population contained on atypical histopathology biopsy slide.

[0012] A typical tissue biopsy sample consists of a 5 to 10 micron sliceof tissue that is placed on a glass microscope slide using techniqueswell known in the field of pathology. This tissue slice is a crosssection of the body organ that is being studied. The tissue consists ofa variety of different types of cells. Often a pathologist desires toremove only a small portion of the tissue for further analysis.

[0013] LCM employs a thermoplastic transfer film that is placed on topof the tissue sample. This film is manufactured containing organic dyesthat are chosen to selectively absorb in the near infrared region of thespectrum overlapping the emission region of common AlGaAs laser diodes.When the film is exposed to the focused laser beam the exposed region isheated by the laser and melts, adhering to the tissue in the region thatwas exposed. The film is then lifted from the tissue and the selectedportion of the tissue is removed with the film.

[0014] Thermoplastic transfer films such as a 100 micron thick ethylvinyl acetate (EVA) film available from Electroseal Corporation ofPompton Lakes, N.J. (type E540) have been used in LCM applications. Thefilm is chosen to have a low melting point of about 90° C.

[0015] The thermoplastic EVA films used in LCM techniques have beendoped with dyes, such as an infrared napthalocyanine dye, available fromAldrich Chemical Company (dye number 43296-2 or 39317-7). These dyeshave a strong absorption in the 800 nm region, a wavelength region thatoverlaps with laser emitters used to selectively melt the film. The dyeis mixed with the melted bulk plastic at an elevated temperature. Thedyed plastic is then manufactured into a film using standard filmmanufacturing techniques. The dye concentration in the plastic is about0.001 M.

[0016] While the films employed in LCM applications have provedsatisfactory for the task, they have several drawbacks. The opticalabsorption of a dye impregnated film is a function of its thickness.This property of the film may be in conflict with a desire to selectfilm thickness for other reasons.

[0017] The organic dyes which are used to alter the absorptioncharacteristics of the films may have detrimental photochemistry effectsin some cases. This could result in contamination of LCM samples. Inaddition, the organic dyes employed to date are sensitive to thewavelength of the incident laser light and thus the film must be matchedto the laser employed.

SUMMARY OF THE INVENTION

[0018] There is a particular need for an instrument that is well suitedfor laser capture microdissection. There is also a particular need foran improved method of laser capture microdissection.

[0019] A first aspect of the invention is implemented in an embodimentthat is based on a laser capture microdissection method, comprising:providing a sample that is to undergo laser capture microdissection;positioning said sample within an optical axis of a laser capturemicrodissection instrument; providing a transfer film carrier having asubstrate surface and a laser capture microdissection transfer filmcoupled to said substrate surface; placing said laser capturemicrodissection transfer film in juxtaposition with said sample with apressure sufficient to allow laser capture microdissection transfer of aportion of said sample to said laser capture microdissection transferfilm, without forcing nonspecific transfer of a remainder of said sampleto said laser capture microdisection film; and then transferring aportion of said sample to said laser capture microdissection transferfilm, without forcing nonspecific transfer of a remainder of said sampleto said laser capture microdissection transfer film.

[0020] A second aspect of the invention is implemented in an embodimentthat is based on a laser capture microdissection instrument, comprising:an inverted microscope including: an illumination/laser opticalsubsystem; a translation stage coupled to said illuminator/laser opticalsubsystem; a cap handling subsystem coupled to said translation stage; avacuum chuck subsystem coupled to said translation stage; and a manualjoystick subsystem coupled to said translation stage.

[0021] These, and other, aspects and objects of the invention will bebetter appreciated and understood when considered in conjunction withthe following description and the accompanying drawings. It should beunderstood, however, that the following description, while indicatingpreferred embodiments of the invention and numerous specific detailsthereof, is given by way of illustration and not of limitation. Manychanges and modifications may be made within the scope of the inventionwithout departing from the spirit thereof, and the invention includesall such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] A clear conception of the advantages and features constitutingthe invention, and of the components and operation of model systemsprovided with the invention, will become more readily apparent byreferring to the exemplary, and therefore nonlimiting, embodimentsillustrated in the drawings accompanying and forming a part of thisspecification, wherein like reference numerals (if they occur in morethan one view) designate the same elements. Consequently, the claims areto be given the broadest interpretation that is consistent with thespecification and the drawings. It should be noted that the featuresillustrated in the drawings are not necessarily drawn to scale.

[0023]FIG. 1 illustrates a perspective view of a laser capturemicrodissection inverted microscope, representing an embodiment of theinvention;

[0024] FIGS. 2A-2B illustrate orthographic views of the laser capturemicrodissection (LCM) inverted microscope shown in FIG. 1;

[0025]FIG. 3 illustrates a partial cross-sectional view of an LCMinverted microscope, representing an embodiment of the invention;

[0026]FIG. 4 illustrates a partial cross-sectional view of an LCMinverted microscope, representing an embodiment of the invention;

[0027]FIG. 5 illustrates a cross-sectional view of a cap handlingsubassembly, representing an embodiment of the invention;

[0028]FIG. 6 illustrates an elevational view of a cap handlingsubassembly in a load position, representing an embodiment of theinvention;

[0029]FIG. 7 illustrates a top plan view of the apparatus in theposition depicted in FIG. 6;

[0030]FIG. 8 illustrates an elevational view of a cap handlingsubassembly in an inspect position, representing an embodiment of theinvention;

[0031]FIG. 9 illustrates a top plan view of the apparatus in theposition depicted in FIG. 8;

[0032]FIG. 10 illustrates an elevational view of a cap handlingsubassembly in an unload position, representing an embodiment of theinvention;

[0033]FIG. 11 illustrates a top plan view of the apparatus in theposition depicted in FIG. 10;

[0034]FIG. 12 illustrates a top plan view of a vacuum chuck,representing an embodiment of the invention;

[0035]FIG. 13 illustrates a cross-sectional view of a vacuum chuck,representing an embodiment of the invention;

[0036]FIG. 14 illustrates a schematic diagram of a combined illuminationlight/laser beam delivery system, representing an embodiment of theinvention;

[0037]FIG. 15 illustrates a schematic view of a combinedillumination/laser beam delivery system with a diffuser in place,representing an embodiment of the invention;

[0038]FIG. 16 illustrates a schematic view of a combinedillumination/laser beam delivery system with a cap in place,representing an embodiment of the invention;

[0039]FIG. 17 illustrates a schematic view of an integratedcap/diffuser, representing an embodiment of the invention; and

[0040]FIG. 18 illustrates a schematic view of an integratedcap/diffuser, representing an embodiment of the invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0041] The invention and the various features and advantageous detailsthereof are explained more fully with reference to the nonlimitingembodiments that are illustrated in the accompanying drawings anddetailed in the following description. Descriptions of well knowncomponents and processing techniques are omitted so as not tounnecessarily obscure the invention in detail.

[0042] The entire contents of U.S. Ser. No. 60/037,864, filed Feb. 7,1997 entitled “Laser Capture Microdissection Device,” (Docket No.ARCT-002); U.S. Ser. No. 08/797,026, filed Feb. 7, 1997; U.S. Ser. No.08/800,882, filed Feb. 14, 1997; U.S. Ser. No. 60/060,731, filed Oct. 1,1997; and U.S. Ser. No. 60/060,732, filed Oct. 1, 1997 are herebyexpressly incorporated by reference into the present application as iffully set forth herein.

[0043] Turning to FIG. 1, a perspective view of an inverted microscope100 for laser capture microdissection (LCM) is depicted. The invertedmicroscope 100 includes a variety of subsystems, particularly adaptedfor the LCM technique which combine to provide synergistic andunexpectedly good results. In alternative embodiments, the microscopedoes not need to be an inverted microscope.

[0044] A cap handling mechanic subassembly 110 provides structure forpicking a microcentrifuge tube cap 120 from a supply 122 and placing themicrocentrifuge tube cap 120 on top of a sample that is to undergo LCM.In the depicted embodiment, the microcentrifuge tube cap 120 is acylindrical symmetric plastic cap and the supply 122 includes eight ofthe consumables on a dovetail slide 124. In the depicted embodiment,there is a laser capture microdissection transfer film coupled to thebottom of the microcentrifuge tube cap 120. The cap handling mechanicsubassembly 110 is depicted in one of several possible positions whereina working end 112 of the cap handling mechanic subassembly 110 ispositioned in a vial capping station 114. The movement of the caphandling mechanic subassembly 110 will be described in more detailbelow.

[0045] A glass slide 130 upon which the sample to be microdissected islocated and upon which the microcentrifuge tube cap 120 is placed, islocated in the primary optical axis of the inverted microscope 100. Inalternative embodiments, the slide that supports the sample can be madeof other substantially transparent materials, for example, plastics suchas polycarbonate. The glass slide 130 is supported and held in place bya vacuum chuck 140. The vacuum chuck 140 is a substantially flat surfacethat engages the glass slide 130 through a manifold (not shown) so as tohold the glass slide 130 in place while the microcentrifuge tube cap 120is picked and placed and while a translation stage 145 is manipulated inan X-Y plane. In alternative embodiments, the translation stage can beconfigured so as to have the capability of being moved along a Z axis.

[0046] The translation stage 145 can be manipulated using a pair ofrotary controls (not shown in FIG. 1). In addition, the translationstage 145 can be manipulated using a joystick 150. The joystick 150 isconnected to the translation stage 145 through a spherical mounting 152and a bracket 154. The joystick 150 includes a second spherical mounting156 within a static bracket 158. The joystick provides simultaneous Xand Y movement. Further, this simultaneous movement can be effected within a single handed manner. The acquisition of samples is thus madequicker.

[0047] Mechanical leverage is provided by the fact that the lengthbetween the spherical mounting 152 and the second spherical mounting 156is less than the length between the second spherical mounting 156 andthe bottom end of the joystick 150. This leverage ratio is not neededfor multiplication of force, but for the reduction in scalar movement.This ratio should be less than 1/5, preferably approximately 1/7. Thisratio can be adjusted to provide the optimal resolution needed in termsof sample movement as a function of operator hand movement.

[0048] In addition, the joystick provides tactile feedback not availablewith electronic controls or geared linkages. The joystick 150 permitssimultaneous movement of the translation stage 145 in two directions (Xand Y) as a function of a single vector movement of an operator's hand.This important feature provides an unexpected result in that the speedwith which features to be microdissected can be positioned in theprincipal optical axis of the inverted microscope 100 is significantlyincreased.

[0049] Still referring to FIG. 1, the inverted microscope 100 includesan LCM optical train 160. The LCM optical train 160 is mounted on anillumination arm 165. A white light illuminator 170 is also mounted onthe illumination arm 165. White light from the illuminator 170 passesdownward toward the microcentrifuge tube cap 120 through a dichroicmirror 180 and a focusing lens 190. A laser diode 175 with collimatingoptics emits a beam 177 that is reflected by a beam steering mirror 185.After the beam 177 is reflected by the beam steering mirror 185 it isincident upon the dichroic mirror 180. The dichroic mirror 180 is adichroic that reflects the beam 170 downward through the focusing lens190 toward the microcentrifuge tube cap 120. Simultaneously, thedichroic mirror 180 allows white light from the illuminator 170 to passdirectly down through the focusing lens 190 toward the microcentrifugetube cap 120. Thus, the beam 177 and the white light illumination aresuperimposed. The focusing lens 190 also adjusts the beam spot size.

[0050] Turning now to FIGS. 2A-2B, two orthographic views of theapparatus depicted in FIG. 1 are illustrated. A white light illuminationpath 210 and a laser beam path 220 can be seen in both FIGS. 2A and 2B.It can be appreciated from FIG. 2A that both of the paths includedelivery of optical information to an image acquisition system 230.Similarly, it can be appreciated from FIG. 2B that the illumination beampath includes delivery of optical information to a binocular set 240. Inalternative embodiments, the eyepiece assembly (i.e., ocular) caninclude a monocular.

[0051] Turning to FIG. 3, a block schematic diagram of an optical trainaccording to the invention is depicted. A laser beam path 310 begins ata film activation laser 320. The laser beam path 310 is then reflectedby a mirror 330. The laser beam path 310 is then reflected by a dichroicmirror 340. The laser beam path 310 is then focused by a lens 350. Thelens 350 can optionally be associated with structure for changing thebeam diameter such as, for example, a variable aperture. The laser beampath 310 then passes downward toward the microcentrifuge tube cap 120.The laser beam path 310 then passes through an objective lens 360 and isthen reflected. A cut-off filter 390 is installed in the ocular 370. Thecut-off filter 390 can reflect and/or absorb the energy from the laserbeam.

[0052] The position of the laser beam path 310 with respect to theportion of the sample that is to be acquired by the microcentrifuge tubecap 120 can be seen by an operator via the image acquisition system 230(not shown in FIG. 3), which can include a camera. In idle mode, thelaser beam path 310 provides a visible low amplitude signal that can bedetected via the acquisition system 230. In pulse mode, the laser beampath 310 delivers energy to the microcentrifuge tube cap 120 and theoptical characteristics of the cut-off filter 390 attenuate the laserbeam path 310 sufficiently so that substantially none of the energy fromthe laser beam exits through ocular 370.

[0053] Suitable laser pulse widths are from 0 to approximately 1 second,preferably from 0 to approximately 100 milliseconds, more preferablyapproximately 50 milliseconds. In a preferred embodiment the wavelengthof the laser is 810 nannometers. In a preferred embodiment the spot sizeof the laser at the EVA material located on microcentrifuge tube cap 120is variable from 0.1 to 100 microns, preferably from 1 to 60 microns,more preferably from 5 to 30 microns. These ranges are relativelypreferred when designing the optical subsystem. From the standpoint ofthe clinical operator, the widest spot size range is the most versatile.A lower end point in the spot size range on the order of 5 microns isuseful for transferring single cells.

[0054] Suitable lasers can be selected from a wide power range. Forexample, a 100 watt laser can be used. On the other hand, a 50 mW lasercan be used. The laser can be connected to the rest of the opticalsubsystem with a fiber optical coupling. Smaller spot sizes areobtainable using diffraction limited laser diodes and/or single modefiber optics. Single mode fiber allows a diffraction limited beam.

[0055] While the laser diode can be run in a standard mode such asTEM₀₀, other intensity profiles can be used for different types ofapplications. Further, the beam diameter could be changed with a steppedlens instead of lens 350.

[0056] Changing the beam diameter permits the size of the portion of thesample that is acquired to be adjusted. Given a tightly focused initialcondition, the beam size can be increased by defocusing. Given adefocused initial condition, the beam size can be decreased by focusing.The change in focus can be in fixed amounts. The change in focus can beobtained by means of indents on a movable lens mounting and/or by meansof optical glass steps. In any event, increasing/decreasing the opticalpath length is the effect that is needed to alter the focus of the beam,thereby altering the spot size. For example, inserting a stepped glassprism 380 into the beam so the beam strikes one step tread will changethe optical path length and alter the spot size.

[0057] Turning now to FIG. 4, a schematic block diagram of anotherembodiment of an instrument according to the invention is depicted. Inthis embodiment, a light source 410 (e.g., fluorescence laser), emits aspecific wavelength or wavelength range. The specific wavelength orwavelength range of a beam 420 emitted by the light source 410 ischosen, or filtered, to excite a fluorescent system (e.g., chemicalmarkers and optical filtering techniques that are known in the industry)that is incorporated in or applied to the sample to be microdissected.The frequency of a beam 420 emitted by the fluorescence laser 410 can betuned. The sample includes at least one member selected from the groupconsisting of chromophores and fluorescent dyes (synthetic or organic),and, the process of operating the instrument includes identifying atleast a portion of said sample with light that excites the at least onemember, before the step of transferring said portion of said sample tosaid laser capture microdissection transfer film.

[0058] Still referring to FIG. 4, the beam 420 is reflected by a mirror430. The beam 420 is then reflected by the dichroic mirror 340. In thisway the beam 420 can be made coincident with both the laser beam path310 and the white light from illuminator 170. It should be noted thatthe beam 420 and the laser beam path 310 are shown in a spaced-apartconfiguration for clarity only. The beam 420 and the laser beam path 310can be coaxial. Fluorescence emitted by the sample beneath themicrocentrifuge tube cap 120 then travels through the objective lens 360to be viewed by the operator through ocular 370.

[0059] Turning now to FIG. 5, a cross-sectional view of the cap handlingmechanic subassembly 110 is depicted. The cap handling mechanicsubassembly 110 includes a dampener 510. The dampener 510 is a structurefor damping vertical motion of the cap handling mechanic subassembly110. The dampener 510 is adapted to lower the microcentrifuge tube cap120 down towards the translation stage in a reproducible manner. Thedampener 510 can be an air dampener (e.g., pneumatic tube) or liquiddampener (e.g., hydraulic tube) or any other dynamic structure capableor retarding the vertical motion of the subassembly 110 so as not togenerate an impulse. As the microcentrifuge tube cap 120 contacts theslide on which the sample rests (not shown), the working end 112 of anarm 520 that is coupled to the dampener 510 continues downward at areproducible rate. Therefore, the top of the microcentrifuge tube cap120 rises relative to the bottom of a weight 530. It can be appreciatedthat the cap 120 contacts the slide, before the weight 530 contacts thecap 120. In this way, the microcentrifuge tube cap 120 undergoes aself-leveling step before it is contacted and pressed against the slideby weight 530. As the weight 530 contacts the microcentrifuge tube cap120 the working end 112 of arm 520 continues along its downward path.Therefore, the application of the weight 530 to microcentrifuge tube cap120 is also a self-leveling step. By controlling the mass of weight 530,the force per unit area between the bottom of the microcentrifuge tubecap 120 and the slide can be controlled. After the sample on the slidehas undergone LCM, the arm 520 can be raised. By raising the arm, theweight 530 is first picked off the microcentrifuge tube cap 120 and thenthe microcentrifuge tube cap 120 is picked up off of the slide. Thedampener within the mechanism acts as a dash pot to control the velocityof the pickup arm.

[0060] The position of the translation stage is independent relative tothe position of the cap handling mechanic subassembly 110. Theserelative positions can be controlled by the pair of rotary controls 147.It is to be noted that the pair of rotary controls 147 are depicted withtheir axes parallel to the axis of the microcentrifuge tube cap 120 inFIG. 5. However, the pair of rotary controls 147 can be configured inany orientation through the use of mechanical linkages such as gears.

[0061] Turning now to FIG. 6, the cap handling mechanic subassembly 110is depicted in a load position. In the load position, the working end112 of the arm 520 is located directly over the dovetail slide 124. Inthis position, the working end 112 grasps a microcentrifuge tube cap120. After grasping the microcentrifuge tube cap 120, the arm 520 israised, thereby picking the microcentrifuge tube cap 120 up.

[0062] Turning now to FIG. 7, a top plan view of the cap handlingmechanic subassembly 110 in the load position can be seen. Before thearm 520 is swung into the load position, a fresh microcentrifuge tubecap is located beneath the axis of the working end 112. After the arm520 is swung clockwise toward the vacuum chuck 140, the caps on dovetailslide 124 will be advanced so as to position a fresh microcentrifugetube cap in place for the next cycle.

[0063] Turning now to FIG. 8, the cap handling mechanic subassembly 110is depicted in an inspect position. When positioned in the inspectposition, the working end 112 of the arm 520 is located coincident withthe principal optical axis of the instrument. This is the position inwhich the arm 520 is lowered to permit first the self-leveling of themicrocentrifuge tube cap 120 and then the self-leveling of the weight530 on top of the microcentrifuge tube cap 120. After LCM, the arm 520is raised in this position to put the weight 530 off the microcentrifugetube cap 120 and then the microcentrifuge tube cap 120 off the slide(not shown).

[0064] The weight 530 is a free floating weight so that when it is seton top of the cap, the cap and weight are free to settle. The freefloating weight permits the even application of pressure. For example, aweight of 30 grams can be used in the case where the total surface areaof the laser capture microdissection transfer film is approximately 0.26square inches.

[0065] Referring now to FIG. 9, a top plan view of the cap handlingmechanic subassembly 110 in the inspect position is depicted. It can beappreciated from this view that the working end 112 of the arm 520 islocated above the glass slide 130.

[0066] Turning now to FIG. 10, the cap handling mechanic subassembly 110is depicted in an unload position. In the unload position, the workingend 112 of the arm 520 and the cap 120 (aka consumable) with the LCMattached tissue are all located above the vial capping station 114.After being positioned on axis with the vial capping station 114, themicrocentrifuge tube cap 120 is lowered directly down onto, and into, ananalysis container 1000. After the microcentrifuge tube cap 120 isinserted into the analysis container 1000, the working end 112 of thearm 520 is raised up. The working end 112 of the arm 520 is then rotatedin a clockwise direction until it is above a fresh consumable(corresponding to the position depicted in FIGS. 6-7).

[0067] Turning now to FIG. 11, a top plan view of the cap handlingmechanic subassembly 110 in the unload position is depicted. In thisposition, the arm 520 is positioned away from the vacuum chuck 140. Theanalysis container 1000 (not visible in FIG. 11) is pushed upward so asto engage the microcentrifuge tube cap 120 (not visible in FIG. 11). Theresultant sealed analysis container 1000 is then allowed to free fallback into a supporting bracket 1010 (see FIG. 10). The sealed analysiscontainer 1000 together with the microcentrifuge tube cap 120 can thenbe taken from the bracket 1010 either manually or automatically.

[0068] Turning now to FIG. 12, a top plan view of the vacuum chuck 140is depicted. A top surface 1210 of the vacuum chuck 140 includes a firstmanifold hole 1020 and a second manifold hole 1030. In alternativeembodiments, there can be any number of manifold holes. The vacuum chuck140 includes a beam path hole 1040. When the instrument is in operation,the glass slide (not shown), or other sample holder, is placed over thebeam path hole 1040 and the manifold holes 1020-1030. After the glassslide is placed in position, a vacuum is pulled through a manifoldconnected to the holes 1020-1030, thereby holding the glass slide inposition over the beam path hole 1040. Although a medium or even a highvacuum can be applied, a low vacuum is sufficient to hold the glassslide in place during the LCM process. A sufficient vacuum can even begenerated with an inexpensive aquarium pump run in reverse.

[0069] The holding force exerted on the glass slide 130 is a function ofthe applied vacuum, the size and shape of the manifold holes 1020-1030and the spacing between the top surface of the translation stage and thebottom surface of the glass slide 130. The spacing between thetranslation stage and the glass slide 130 is a function of the flatnessof the surfaces and the elasticity of the corresponding structures.

[0070] The level of vacuum is adjusted so that the glass slide 130, orother sample carrier, can be translated with regard to the translationstage. This translation capability is present when the vacuum is off andwhen the vacuum is on.

[0071] There is some leakage around the perimeter of the glass slide 130which modulates the force holding the glass slide 130 in place.Accordingly, a moderate force (e.g., 5 pounds) applied to the edge ofthe glass slide is sufficient to cause movement of the glass slide 130with regard to the translation stage when the vacuum is engaged.

[0072] Turning now to FIG. 13, a cross section of the vacuum chuck isdepicted with a glass slide 130 in place. The vacuum that holds theglass slide 130 in place is pulled through conduit 1320. The conduit1320 is connected to a circular manifold 1310. The circular manifold1310 is coupled with the manifold holes 1020-1030.

[0073] It can be appreciated from FIG. 13 that there are no pins, orother structures, that project above the top surface of the vacuum chuck140. This permits the glass slide 130 to be moved in any directionparallel with the top surface without constraint.

[0074] Turning now to FIG. 14, a very high numerical apertureilluminator 1400 for an LCM device is depicted. The illuminator 1400provides a large working distance. A fiber optic 1410 provides a sourceof white light illumination. The diverging beam 1420 from the fiberoptic 1410 can have a numerical aperture of approximately 0.4. Acollimator lens 1430 collimates the light from the fiber optic 1410. Thecollimator lens 1430 can be an aspheric lens (e.g., a Melles Griot (01LAG 025) aspheric-like lens).

[0075] A collimated beam 1440 from the collimator lens 1430 then passesthough a beam splitter 1450. The beam splitter 1450 permits theinjection of a laser beam 1460. After reflection by the beam splitter1450, the laser beam 1460 is coaxial with the white light illumination.Both types of light then reach a condenser lens 1470. Condenser lens1470 can be a Melles Griot (01 LAG 010) or (01 LAG 010) or other similaraspheric-like lens. The condensed coaxial beams are then incident uponand pass through the microcentrifuge tube cap 120. The focusing beamthat results from the condenser lens 1470 can have a numerical apertureof approximately 0.8. This can be characterized as a focusing beam. Themicrocentrifuge tube cap 120 is located on top of a slide with cells tobe sampled (not shown).

[0076] Turning now to FIG. 15, another embodiment of the high numericalaperture illuminator is depicted. In this embodiment, a diffuser 1500 islocated beneath the condenser lens 1470 at above the glass slide 130that contains the cells to be sampled.

[0077] More generally, any suitable scattering media can be used toprovide the functions of the diffuser 1500. Providing such a scatteringmedia near the tissue to scatter the light results in dramaticallyimproved illumination of the sample and much better visualization. Ascattering media of this type eliminates the need for refractive indexmatching of the sample. Such a scattering media can allow visualizationof the cell nucleus and other subcellular structures that would normallybe obscured by normal illumination techniques.

[0078] The scattering media can be a diffuser material. A diffusermaterial that is suitable for use as the scattering media is milk oropal glass which is a very dense, fine diffuser material. For instance,a suitable milk glass is available from Edmund Scientific as Part No.P43,717. Standard laser printer/photocopier paper can even be used asthe scattering media. Other types of transparent scattering media can beused, such as, for example, frosted glass, a lenticular sheet, a volumediffuser, and/or a surface diffuser. In any event, the scattering mediashould be a material that aggressively scatters the illumination light.A single sheet of typical ground glass is generally inadequate and needsto be combined in multiple layers as a serial stack of three or foursheets of ground glass to diffuse the illumination light sufficiently.

[0079] Turning now to FIG. 16, after the diffuser 1500 is replaced witha microcentrifuge tube cap 120, the desired cells can be located usingthe image acquired during the step represented in FIG. 15. Then, thelaser beam 1460 can be introduced, reflected off the beam splitter 1450and directed into the microcentrifuge tube cap 120 so as to acquire thedesired sample.

[0080] The purpose of the illuminator design is to provide a very highnumerical aperture illuminator for an LCM device. Such an LCM devicerequires a large working distance. While an illuminator that uses a 40×objective with 0.8 numerical aperture may seem to give bettervisualization, this design has problems since the working distance forthe 40× objective is very small, (e.g., less than 1 millimeter). Thus itis critical for a design that uses a thick dome carrier to have anillumination design with a much longer working distance. A thick domecarrier is a sample carrier whose top and bottom are spaced apart morethan a small distance. This is important because the sample is adjacentthe bottom of the sample carrier and the objective cannot move closer tothe sample than the top of the sample carrier.

[0081] The focusing lens 190 can be replaced with a Melles Griotaspheric condenser lens such as a 01 LAG 010. Such a lens has anumerical aperture of about 0.75 and a working distance of about 25millimeters. Such a lens is not corrected for chromatic aberrations likethe 40× objective. Experiments done using a spherical lens as acondenser gave good improvement in visualization. This spherical lensclearly did not have the corrections for aberrations that are built intothe 40× objective.

[0082] The laser beam can be focused through this condenser lens likethe focusing lens 190. This condenser lens has roughly one-half thefocal length of the current lens so the laser beam will be focused downto roughly 15 microns.

[0083] In an alternative embodiment the design could use a compound lenslike the lens in a barcode scanner. Such a compound lens would have acentral region for the laser and a surrounding region that would act asa high numerical aperture with regard to the white light illumination.

[0084] Turning now to FIG. 17, in one embodiment the diffuser 1500 canbe located adjacent to the microcentrifuge tube cap 120. In thisembodiment the microcentrifuge tube cap 120 is located just above theglass slide 130. Collimated light 1700 is incident upon the diffuser1500. The diffuser 1500 causes the collimated light to enter into andpass through the cap at an infinite variety of angles. In this way,shadows are reduced and the quality of the imagery is improved.

[0085] The diffuser 1500 can be a volumetric diffuser or a surfacediffuser. In the case of a volumetric diffuser, the diffuser 1500 can befrosted glass, a speckle based holographic diffuser or even a piece ofpaper. In the case of a surface diffuser, the diffuser 1500 can be alenticular sheet, a speckle based holographic surface diffuser or anyother suitable topological surface.

[0086] Turning now to FIG. 18, the diffuser 1500 in this embodiment islocated adjacent to the bottom of the microcentrifuge tube cap 120. Thecollimated light 1700 passes through the microcentrifuge tube cap 120and is incident upon the diffuser 1500. As the previously collimatedlight emerges from the diffuser 1500 it is scattered into a wide rangeof angles. In this embodiment, the diffuser 1500 is spaced apart fromthe glass slide 130.

[0087] The scattering media (e.g., diffuser 1500) can be directly orindirectly connected to the transfer film carrier and/or the LCMtransfer film. Alternatively, the scattering media can be formed on asurface of, or the interior of, the transfer film carrier and/or the LCMtransfer film. The scattering media can be fabricated so as to shape theLCM beam and/or the illumination beam. The scattering media needs to bewithin a few millimeters of the sample to be effective. A fewmillimeters means less than one centimeter, preferably less than fivemillimeters.

[0088] The process of operating the instrument begins by visualizing thetissue from which the sample is to be acquired. The tissue is then movedto bring the portion that is to be acquired directly below the principalaxis of the instrument. A laser capture microdissection transfer film isthen set over the desired area. In a preferred embodiment the film isspaced to within a few microns of the top surface of the sample.Alternatively, the film can be placed in contact with the top of thesample with a pressure sufficient to allow transfer without forcingnonspecific transfer. Finally, the laser is pulsed to heat the film andremove the tissue. The film needs to be pulled off of the samplequickly. Though the velocity should be such that the sample isthixotropically sheared.

[0089] Practical Applications of the Invention

[0090] A practical application of the invention that has value withinthe technological arts is the collection of a large database of geneexpression patterns of both healthy and diseased tissue, at differentstages of diseases. This database will be used to more fully understandthat pathogenesis of cancer and infectious diseases. The invention willenable a scientist to identify gene patterns and incorporate thisinformation into effective diagnostics for disease. The invention willallow medical doctors to compare actual patient tissue samples witharchived data from patient samples at different disease stages, therebyallowing them to prescribe more effective stage therapies, eliminateunnecessary procedures, and reduce patient suffering. Other researchareas where the invention will find use are drug discovery,developmental biology, forensics, botany, and the study of infectiousdiseases such a drug-resistant tuberculosis. There are virtuallyinnumerable uses for the invention, all of which need not be detailedhere.

[0091] Advantages of the Invention

[0092] A laser capture microdisection instrument and/or methodrepresenting an embodiment of the invention can be cost effective andadvantageous for at least the following reasons. The invention willreplace current methods with better technology that allows for moreaccurate and reproducible results. The invention can be used to providea low cost injection molded polymer disposable that integrates a lasercapture microdissection transfer film into the interior surface of ananalysis container such as a microcentrifuge tube (e.g., an EPPENDORF™tube).

[0093] All publications, patent applications, and issued patentsmentioned in this application are hereby incorporated herein byreference in their entirety to the same extent as if each individualpublication, application, or patent was specifically and individuallyindicated to be incorporated in its entirety by reference.

[0094] All the disclosed embodiments of the invention described hereincan be realized and practiced without undue experimentation. Althoughthe best mode of carrying out the invention contemplated by theinventors is disclosed above, practice of the invention is not limitedthereto. It will be manifest that various additions, modifications andrearrangements of the features of the invention may be made withoutdeviating from the spirit and scope of the underlying inventive concept.Accordingly, it will be appreciated by those skilled in the art that theinvention may be practiced otherwise than as specifically describedherein.

[0095] For example, the individual components need not be formed in thedisclosed shapes, or assembled in the disclosed configuration, but couldbe provided in virtually any shape, and assembled in virtually anyconfiguration. Further, the individual components need not be fabricatedfrom the disclosed materials, but could be fabricated from virtually anysuitable materials. Further, although the LCM instrument disclosedherein is described as a physically separate module, it will be manifestthat the LCM instrument may be integrated into other apparatus withwhich it is associated. Furthermore, all the disclosed elements andfeatures of each disclosed embodiment can be combined with, orsubstituted for, the disclosed elements and features of every otherdisclosed embodiment except where such elements or features are mutuallyexclusive.

[0096] It will be manifest that various additions, modifications andrearrangements of the features of the invention may be made withoutdeviating from the spirit and scope of the underlying inventive concept.It is intended that the scope of the invention as defined by theappended claims and their equivalents cover all such additions,modifications, and rearrangements. The appended claims are not to beinterpreted as including means-plus-function limitations, unless such alimitation is explicitly recited in a given claim using the phrase“means-for.” Expedient embodiments of the invention are differentiatedby the appended subclaims.

What is claimed is:
 1. A laser capture microdissection method,comprising: providing a sample that is to undergo laser capturemicrodissection; positioning said sample on a translation stage of alaser capture microdissection instrument and within an optical axis ofsaid laser capture microdissection instrument; providing a transfer filmcarrier having a substrate surface and a laser capture microdissectiontransfer film coupled to said substrate surface; placing said lasercapture microdissection transfer film in juxtaposition with said samplewith a pressure sufficient to allow laser capture microdissectiontransfer of a portion of said sample to said laser capturemicrodissection transfer film, without forcing nonspecific transfer of aremainder of said sample to said laser capture microdisection film; andthen moving said sample and said translation stage with a manualjoystick subsystem that is connected to said translation stage; and thentransferring a portion of said sample to said laser capturemicrodissection transfer film, without forcing nonspecific transfer of aremainder of said sample to said laser capture microdissection transferfilm.
 2. The laser capture microdissection method of claim 1, whereinmoving said sample and said translation stage with said manual joysticksubsystem includes simultaneous X and Y movement.
 3. The laser capturemicrodissection method of claim 1, wherein moving said sample and saidtranslation stage with said manual joystick subsystem includes reducinga scalar movement defined by an operator.
 4. A laser capturemicrodissection instrument, comprising: a translation stage; and amanual joystick subsystem coupled to said translation stage.
 5. Thelaser capture microdissection instrument of claim 4, wherein said manualjoystick subsystem includes a joystick that is coupled to saidtranslation stage through a first spherical mounting that is movablyconnected to said joystick and a bracket that is mechanically connectedto both said spherical mounting and said translation stage.
 6. The lasercapture microdissection instrument of claim 5, wherein said manualjoystick subsystem includes a joystick having a second sphericalmounting that is movably connected to a static bracket.
 7. The lasercapture microdissection instrument of claim 6, wherein a first lengthbetween said first spherical mounting and said second spherical mountingis less than a second length between said second spherical mounting anda bottom end of said joystick.
 8. The laser capture microdissectioninstrument of claim 7, wherein a ratio of said first length to saidsecond length is less than 1/5.
 9. The laser capture microdissectioninstrument of claim 8, wherein said ratio is approximately 1/7.
 10. Thelaser capture microdissection instrument of claim 4, further comprisingan illumination/laser optical subsystem.
 11. The laser capturemicrodissection instrument of claim 4, further comprising a transferfilm carrier handling subsystem.
 12. The laser capture microdissectioninstrument of claim 4, further comprising a vacuum chuck subsystemconnected to said translation stage.
 13. An inverted microscope,comprising: a translation stage; and a manual joystick subsystemconnected to said translation stage.
 14. The inverted microscope ofclaim 13, wherein said manual joystick subsystem includes a joystickthat is coupled to said translation stage through a first sphericalmounting that is movably connected to said joystick and a bracket thatis mechanically connected to both said spherical mounting and saidtranslation stage.
 15. The inverted microscope of claim 14, wherein saidmanual joystick subsystem includes a joystick having a second sphericalmounting that is movably connected to a static bracket.
 16. The invertedmicroscope of claim 15, wherein a first length between said firstspherical mounting and said second spherical mounting is less than asecond length between said second spherical mounting and a bottom end ofsaid joystick.
 17. The inverted microscope of claim 16, wherein a ratioof said first length to said second length is less than 1/5.
 18. Theinverted microscope of claim 17, wherein said ratio is approximately1/7.
 19. The inverted microscope of claim 13, further comprising anillumination/laser optical subsystem.
 20. The inverted microscope ofclaim 13, further comprising a transfer film carrier handling subsystem.21. The inverted microscope of claim 13, further comprising a vacuumchuck subsystem connected to said translation stage.